Visible light-induced switching of soft matter materials properties based on thioindigo photoswitches

Thioindigos are visible light responsive photoswitches with excellent spatial control over the conformational change between their trans- and cis- isomers. However, they possess limited solubility in all conventional organic solvents and polymers, hindering their application in soft matter materials. Herein, we introduce a strategy for the covalent insertion of thioindigo units into polymer main chains, enabling thioindigos to function within crosslinked polymeric hydrogels. We overcome their solubility issue by developing a thioindigo bismethacrylate linker able to undergo radical initiated thiol-ene reaction for step-growth polymerization, generating indigo-containing polymers. The optimal wavelength for the reversible trans-/cis- isomerisation of thioindigo was elucidated by constructing a detailed photochemical action plot of their switching efficiencies at a wide range of monochromatic wavelengths. Critically, indigo-containing polymers display significant photoswitching of the materials’ optical and physical properties in organic solvents and water. Furthermore, the photoswitching of thioindigo within crosslinked structures enables visible light induced modulation of the hydrogel stiffness. Both the thioindigo-containing hydrogels and photoswitching processes are non-toxic to cells, thus offering opportunities for advanced applications in soft matter materials and biology-related research.

The manuscript by Loh, Barner-Kowollik, Truong and co-workers reports synthesis and properfies of a polymer comprising thioindigo moiefies in main chains.Especially important is the achievement of the solubility in water.The development of new photoswitchable materials is a vibrant topic, which perfectly fits to the broad readership of Nat.Commun.Nevertheless, I cannot recommend the manuscript for publicafion for the following reasons: 1) The authors claim:"…we pioneer a strategy towards the integrafion of the thioindigo funcfion into polymer main chains, enabling the assessment of the photoswitching of the thioindigo-containing macromolecules…".Actually, there is a publicafion by Irie et al. (hftps://iopscience.iop.org/arficle/10.1143/JJAP.39.L633/meta) describing the incorporafion of thioindigo into the main polymer chains with follow up photoswitching.This study should be cited and compared with the results obtained in the manuscript.
2) The photochemical studies are mainly qualitafive and do not correspond to the current characterizafion standards for photoswitchable molecules.Quantum yields for the forward and backward isomerizafions should be provided for all applied condifions (monomer, polymer in different solvents).
3) The rates of photoisomerizafion and thermal recovery are significantly dependent on the experimental condifions and are, in general, not very helpful.For thermal backwards isomerizafion, Gibbs free acfivafion energies should be obtained, which characterize the thermal barrier of the backward reacfions.These values can be calculated through the Eyring plot.With the Gibbs free acfivafion energies in hand, one can easily esfimate the thermal half-life of the photoinduced species at any temperature.For the publicafion, in addifion to the Gibbs energies, it would be good to provide the values for the thermal half-lifes at 25 C. 4) Although I completely understand the difficulfies regarding the convenfional NMR detecfion of shortliving photoinduced species, nowadays this is not a problem anymore.There is a large amount of developed approaches for real-fime NMR spectroscopy with in situ irradiafion, which allow the detecfion even of short-living photoreacfion intermediates.Please, check the following reviews and references therein: hftps://doi.org/10.1002/cptc.201900109,hftps://doi.org/10.1002/cctc.202201583.Some of these approaches, for example, the one described by Gschwind et al.
(hftps://doi.org/10.1016/j.jmr.2013.04.011), are fast, easy and cheap to implement.Alternafively, mathemafical methods such as the Fisher method (recently used for indigo derivafives hftps://doi.org/10.1002/cptc.201900032)can be applied to calculate the PSS composifions and the spectra of the photoinduced isomers from the absorpfion spectroscopy data.5) Figure 3: I am not sure that the spectra obtained in methanol and water indicate photoswitching because their shapes seem not to change at all.Can some different photochemistry in polymer happen, which simply changes the solubility upon irradiafion?In any case, it would be helpful to perform DFT calculafions of the trans-and cis-forms in corresponding solvents to compare the experimental data with the calculated values of the absorpfion maxima for both forms.6) Although the biocompafibility studies are clear, I did not get, for which purpose such a photoswitchable hydrogel can be applied in medicine.Could the authors add some explanafions to the text?
Reviewer #2 (Remarks to the Author): In their manuscript fitled "Visible light-enabled switching of soft material properfies based on thioindigo photoswitches", Walden et al. react methacrylate-tethered thioindigos with PEG-SH chains and later form crosslinked systems out of these linear polymers, ending up with networks that can be gelated with water.They demonstrate that PEG-thioindigo linear polymers can be photoisomerised in a range of solvents and, when crosslinked, as part of a hydrogel material.In the lafter case, the mechanical properfies of the hydrogel can be tuned by isomerising the switch from trans to cis or vice versa with green and blue light, respecfively.
The authors have done a commendable work in synthesising the thioindigo polymer and in the following material fabricafion.The synthesis seems robust and straighfforward, and the characterisafion of the material has been extended to biocompafibility, which is something most manuscripts do not address in such detail.The manuscript is quite easy to read, and the figures are aesthefically pleasing.There is substanfial novelty as well, seeing that this is the first fime thioindigos (or to our knowledge any indigoids, for that mafter) have been used in this context.However, we feel that the authors have not demonstrated a big enough progress in the field to warrant publicafion in Nature Communicafions: certain limitafions seem to exist, and comparison to already exisfing similar systems is lacking.Thus, we believe this work would be befter suited for a more specialised journal focusing on funcfional materials.
Independent of where the work is published, we feel that it could be greatly improved with the following addifions and modificafions: -In both the introducfion and the discussion/conclusions part, comparison to other photoswitches and photoswitchable hydrogel systems is inadequate.The authors correctly idenfify the rigidity of thioindigos as a potenfial advantage over azobenzenes but do not menfion other indigoid photoswitch classes that share the same property while having some extra advantages of their own.For instance, hemithioindigos are thermally stable, whereas N-funcfionalised indigos can be switched with red light, even in a solid environment (see the references below).It would be important to give proper context for thioindigos and why they might be befter suitable for this purpose than the other aforemenfioned indigoids.
Similarly, what about already exisfing hydrogel systems based on azobenzene (there are many examples): how do your results compare to them?Did you get more or less tuneability in the mechanical properfies than the state-of-the-art azobenzene systems?hftps://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.201700826hftps://pubs.rsc.org/en/content/arficlelanding/2023/sc/d2sc06790k-The system seems to be really slow, mechanical properfies changing over the course of up to an hour.Do the authors have any hypothesis for why this is so slow?Could some ideas be given for how to overcome this problem?Again, how does this compare to exisfing photoresponsive hydrogel materials?-The photoisomerisafion spectra in Figure 3 only show a clear isosbesfic point in chloroform and DMSO.Thus, it seems that in the other solvents there is either degradafion or perhaps photoinduced aggregafion; something that removes photochromic compounds from the solufion.Will you recover the original spectra in fime (which would rule out degradafion)?Either way, this phenomenon should be discussed and perhaps invesfigated to determine that the switching is actually reversible.
-No spectral analysis has been carried out for the hydrogels.This would, however, be important in order to know to which extent the thioindigo switches inside the hydrogel.It was recently observed that for Nfuncfionalised indigos, the indigo concentrafion changes the switching dynamics quite drasfically, and it would thus be interesfing to know whether a similar phenomenon is observed in your case -or if not, this would be a benefit for thioindigo compared to indigo.It should be rather straighfforward to make thin enough hydrogels that could be studied with UV-vis spectroscopy.Then you could see a) what the PSS is like and b) how fast the thermal back-isomerisafion is, and whether this happens on the same fimescale as the changes in the mechanical properfies or not.
-It seems that the mechanical properfies do not return to the original when switched for the first fime.This is understandable for the two-way photochemical switching (Figure 4a) due to both wavelengths forming PSSs that differ from 100% E or Z, but for the thermal back-isomerisafion (Figure 4b) one would expect the mechanical properfies to return to the original for a truly reversible system.It seems that some irreversible change occurs on the first switching cycle, after which switching is reversible.This should be discussed and perhaps some rafionale given, if possible.
-In the introducfion, you menfion that the lack of conformafional ridigity in azobenzenes arises from the rotafional isomerisafion pathway (as compared to in-plane inversion).However, it does not mafter what the mechanism between the planar trans isomer and the twisted cis isomer is, as the conformafional change is dictated by the ground-state minimum of the cis isomer, which is independent of the pathway via which it is reached.Bear in mind that also (thio)indigos isomerise via rotafion and sfill have rigid, planar structures for both isomers.
-A small note: in text, you menfion that no observable differences are seen in Figure 2c during mulfiple isomerisafion cycles.There is, however, a clearly (although slightly) decreasing trend in the absorbance values, so I would rephrase along the lines of "no major degradafion" or "only slight degradafion".

Reviewer #3 (Remarks to the Author):
The authors report visible light-induced switching of soft mafter materials properfies that the integrafion of the thioindigo funcfion into polymer main chains.The photoswitching of thioindigo within crosslinked structures enables visible light induced modulafion of the hydrogel sfiffness.This material shows excellent cytocompafibility.Their discussion is sound overall, but the authors need to address the quesfions listed below before the publicafion is fully warranted.Comments: 1.The molecule (1) shows excellent reversible trans-/cis-photoswitching (>10 cycles), but why does it show only three changes in the material?Can more fimes?2. According to previous works (Nature nanotechnology 10.2 (2015): 161-165; Nature nanotechnology 12.6 (2017): 540-545), light can drive motor to produce a confinuous unidirecfional out-of-equilibrium rotafion, resulfing in a macroscopic contracfion of the enfire network.The mechanism (Fig. 1) proposed by the authors does not seem to explain the problem befter.According to the mechanism proposed by the authors, this change in shape should be completely reversible, but it does not seem to be (Rheological data).And there is no other data to support this result, such as pictures.

Reviewer #1 (Remarks to the Author):
The manuscript by Loh, Barner-Kowollik, Truong and co-workers reports synthesis and properties of a polymer comprising thioindigo moieties in main chains.Especially important is the achievement of the solubility in water.The development of new photoswitchable materials is a vibrant topic, which perfectly fits to the broad readership of Nat.Commun.Nevertheless, I cannot recommend the manuscript for publication for the following reasons: Response: We thank the reviewer for the kind assessment of our report, and the remark on the importance of new photoswitchable macromolecules that are water-soluble.We have included extensive additional experiments to address the concerns raised in the detailed comments and highlight the significance of our work on the applications of thioindiogo phototoswitch in soft matter materials.
1) The authors claim:"…we pioneer a strategy towards the integration of the thioindigo function into polymer main chains, enabling the assessment of the photoswitching of the thioindigo-containing macromolecules…".
Actually, there is a publication by Irie et al.
(https://iopscience.iop.org/article/10.1143/JJAP.39.L633/meta) describing the incorporation of thioindigo into the main polymer chains with follow up photoswitching.This study should be cited and compared with the results obtained in the manuscript.

Response:
We thank the reviewer for referring to a related report of thioindigo photoswitching in polymer thin film that was overlooked.We have included this reference in the introduction (page 3) and discussion (page 6) of our report.
2) The photochemical studies are mainly qualitative and do not correspond to the current characterization standards for photoswitchable molecules.Quantum yields for the forward and backward isomerizations should be provided for all applied conditions (monomer, polymer in different solvents).
Response: As per the reviewer's suggestion, we have enhanced the photochemical studies to better align with current characterisation standards.In particular, we have replaced the switching rate in Figure 2d with the quantum yield for forward and backward isomerisation.We have also provided individual experimental data of the photoswitching at discrete wavelength in the 370-530 nm wavelength range into the supporting information (Section 1.5) as well as a detailed description of how the quantum yields were determined.
3) The rates of photoisomerization and thermal recovery are significantly dependent on the experimental conditions and are, in general, not very helpful.For thermal backwards isomerization, Gibbs free activation energies should be obtained, which characterize the thermal barrier of the backward reactions.These values can be calculated through the Eyring plot.With the Gibbs free activation energies in hand, one can easily estimate the thermal half-life of the photoinduced species at any temperature.For the publication, in addition to the Gibbs energies, it would be good to provide the values for the thermal half-lifes at 25 C.

Response:
We thank the reviewer for their comment.To obtain a universal data set on the photoisomerization process, we have performed quantum chemical calculations (results are provided on page 7-10 and Fig. 5), obtaining an energy barrier of 0.423 eV for the trans-to cis-transformation and an energy barrier of 0.37 eV for the cis-to trans-conversion.Our calculations further account for the hydrogen bonding energy in protic solvents (such as methanol and water), explaining the lower conversion efficiency in such solvents.As requested, we have also reported the experimentally determined thermal half-life at 24 °C in the revised manuscript.

4)
Although I completely understand the difficulties regarding the conventional NMR detection of shortliving photoinduced species, nowadays this is not a problem anymore.There is a large amount of developed approaches for real-time NMR spectroscopy with in situ irradiation, which allow the detection even of short-living photoreaction intermediates.Please, check the following reviews and references therein: https://doi.org/10.1002/cptc.201900109,https://doi.org/10.1002/cctc.202201583.Some of these approaches, for example, the one described by Gschwind et al.
(https://doi.org/10.1016/j.jmr.2013.04.011), are fast, easy and cheap to implement.Alternatively, mathematical methods such as the Fisher method (recently used for indigo derivatives https://doi.org/10.1002/cptc.201900032)can be applied to calculate the PSS compositions and the spectra of the photoinduced isomers from the absorption spectroscopy data.

Response:
We completely agree with the reviewer that an in situ-NMR is the optimal way to characterise these short-lived species.In fact, we are currently in the process of commissioning a laser-coupled NMR at QUT that is expected to be operational later this year.We disagree, however, that such a system, when executed thoroughly, is neither fast, cheap nor easy to install.The purpose of the NMR measurements in the current work was not to determine the photostationary state, but rather to confirm the structure of the isomers and that no degradation or side products were being formed during irradiation.We acknowledge that our reporting of these results was not ideal and have clarified the relevant text in the manuscript.Response: Based on the suggestion of the reviewer, we have conducted quantum chemical calculations of thioindigo photoswitching.These calculations indicate that the hydrogen bonding energy in protic solvents significantly affects the relative Gibbs free energy of both the trans-and cis-isomers, consequently altering their λ abs .Based on our calculations, we also find that the cis-form is more polar than the trans-form.To confirm that photoswitching does occur, we have performed up to 10 cycles of alternating irradiation of green and blue light on the polymer solutions in methanol and water.Our results (Fig. S14) indicate repeatable photoswitching between the cis-and trans-states and no significant photodegradation occurring during light irradiations.

6)
Although the biocompatibility studies are clear, I did not get, for which purpose such a photoswitchable hydrogel can be applied in medicine.Could the authors add some explanations to the text?
Response: As demonstrated in our study, the hydrogels are biocompatible and can be used as a scaffold for the study of dynamic cell-materials interaction i.e., mechanotransduction of cells and modelling of disease progression.Indeed, we observed different cell morphologies when the hydrogel substrate was softened by green light irradiation, compared to cells grown on stiff hydrogels with no green light treatment.To reduce ambiguity, we have removed the 'medicinal' context and included additional texts in the discussion to explain the potential of our photoswitchable hydrogels in cells materials interaction and disease modelling.

Reviewer #2 (Remarks to the Author):
In their manuscript titled "Visible light-enabled switching of soft material properties based on thioindigo photoswitches", Walden et al. react methacrylate-tethered thioindigos with PEG-SH chains and later form crosslinked systems out of these linear polymers, ending up with networks that can be gelated with water.They demonstrate that PEG-thioindigo linear polymers can be photoisomerised in a range of solvents and, when crosslinked, as part of a hydrogel material.In the latter case, the mechanical properties of the hydrogel can be tuned by isomerising the switch from trans to cis or vice versa with green and blue light, respectively.
The authors have done a commendable work in synthesising the thioindigo polymer and in the following material fabrication.The synthesis seems robust and straightforward, and the characterisation of the material has been extended to biocompatibility, which is something most manuscripts do not address in such detail.The manuscript is quite easy to read, and the figures are aesthetically pleasing.There is substantial novelty as well, seeing that this is the first time thioindigos (or to our knowledge any indigoids, for that matter) have been used in this context.However, we feel that the authors have not demonstrated a big enough progress in the field to warrant publication in Nature Communications: certain limitations seem to exist, and comparison to already existing similar systems is lacking.Thus, we believe this work would be better suited for a more specialised journal focusing on functional materials.
Independent of where the work is published, we feel that it could be greatly improved with the following additions and modifications.

Response:
We thank the reviewer for the kind assessment regarding the novelty of our research, and the suggestions to improve our report.Response: As suggested, we have included the references on indigos and hemithioindigos in the introduction with relevant texts, as well as the comparison of our results with azobenzene-containing hydrogels in the Results section.
-The system seems to be really slow, mechanical properties changing over the course of up to an hour.
Do the authors have any hypothesis for why this is so slow?Could some ideas be given for how to overcome this problem?Again, how does this compare to existing photoresponsive hydrogel materials?
Response: The time required for the tuning of mechanical properties relies on several factors.The first is the isomerisation rate, which we have already shown in Figure 2 to be reasonably efficient.The second is the penetration of light throughout the material volume.The molar absorptivity of both the transand cis-isomers is high (close to 10 4 L mol -1 cm -1 ) and so is the thioindigo incorporation into the hydrogel (close to5.5 wt%).These two factors combine to significantly limit the light penetration (and hence mechanical tuning) throughout the material.We have made efforts to minimise the impact of these effects by irradiating with wavelengths offset from the absorption maxima.Further increases in the mechanical tuning rates are expected to be possible by selecting irradiation sources even further removed from the absorption maxima.In addition, as indicated by our chemical quantum calculations, the photoswictching is highly constrained in protic solvents -especially water -which explains the slow photoswitching kinetics of the polymer network.We have included the explanation and relevant -The photoisomerisation spectra in Figure 3 only show a clear isosbestic point in chloroform and DMSO.
Thus, it seems that in the other solvents there is either degradation or perhaps photoinduced aggregation; something that removes photochromic compounds from the solution.Will you recover the original spectra in time (which would rule out degradation)?Either way, this phenomenon should be discussed and perhaps investigated to determine that the switching is actually reversible.

Response:
We thank the reviewer for the feedback on the photoisomerization spectra.We agree the decrease in the 540 nm absorbance band is due to photoinduced aggregation of the polymer chains.
After green/blue light treatment the spectra did not revert to original spectra in all polar solvents, even with prolonged blue light exposure.To rule out any photodegradation effects, we carried out green/blue light irradiations for up to 10 cycles on the polymer solutions in MeOH and water.The results (Fig. S14) show that photoswitching can be induced by green/blue light and no significant photodegradation occurring during the light irradiation processes.
-No spectral analysis has been carried out for the hydrogels.This would, however, be important in order to know to which extent the thioindigo switches inside the hydrogel.It was recently observed that for N-functionalised indigos, the indigo concentration changes the switching dynamics quite drastically, and it would thus be interesting to know whether a similar phenomenon is observed in your case -or if not, this would be a benefit for thioindigo compared to indigo.It should be rather straightforward to make thin enough hydrogels that could be studied with UV-vis spectroscopy.Then you could see a) what the PSS is like and b) how fast the thermal back-isomerisation is, and whether this happens on the same timescale as the changes in the mechanical properties or not.

Response:
We thank the reviewer for the feedback.We have thus undertaken the measurement of the UV-Vis absorbance of hydrogel GelT5 in a cuvette as suggested and observed the photoswitching similar to the changes observed in solution.We have also included the comparison with other solid polymer systems that contain thioindigo or indigo (ref 40: Japanese Journal of Applied Physics 2000, 39 (6B), L633).
-It seems that the mechanical properties do not return to the original when switched for the first time.
This is understandable for the two-way photochemical switching (Figure 4a) due to both wavelengths forming PSSs that differ from 100% E or Z, but for the thermal back-isomerisation (Figure 4b) one would expect the mechanical properties to return to the original for a truly reversible system.It seems that some irreversible change occurs on the first switching cycle, after which switching is reversible.This should be discussed and perhaps some rationale given, if possible.

Response:
We thank the reviewer for their insight.Indeed, the hydrogel stiffness did not reverse to the original value, possibly due to the stabilizing effect of solvent on the trans-isomer.Of note, similar phenomenon was also observed in azobenzene-containing hydrogels (Biomacromolecules 2015, 16,

Figure 3 :
I am not sure that the spectra obtained in methanol and water indicate photoswitching because their shapes seem not to change at all.Can some different photochemistry in polymer happen, which simply changes the solubility upon irradiation?In any case, it would be helpful to perform DFT calculations of the trans-and cis-forms in corresponding solvents to compare the experimental data with the calculated values of the absorption maxima for both forms.
We hope the additional experiments and quantum chemical communications offer considerable insight into the systems, and new progresses that are suitable for publication in Nature Communications.In both the introduction and the discussion/conclusions part, comparison to other photoswitches and photoswitchable hydrogel systems is inadequate.The authors correctly identify the rigidity of thioindigos as a potential advantage over azobenzenes but do not mention other indigoid photoswitch classes that share the same property while having some extra advantages of their own.For instance, hemithioindigos are thermally stable, whereas N-functionalised indigos can be switched with red light, even in a solid environment (see the references below).It would be important to give proper context for thioindigos and why they might be better suitable for this purpose than the other aforementioned indigoids.Similarly, what about already existing hydrogel systems based on azobenzene (there are many examples): how do your results compare to them?Did you get more or less tuneability in the mechanical properties than the state-of-the-art azobenzene systems?